View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector journal of food and drug analysis 23 (2015) 294e302 Available online at www.sciencedirect.com ScienceDirect journal homepage: www.jfda-online.com Original Article Antioxidant activities and contents of flavonoids and phenolic acids of Talinum triangulare extracts and their immunomodulatory effects Dung Y. Liao a, Yu C. Chai b, Sue H. Wang c, Chih W. Chen b, * Ming S. Tsai a, a Department of Bioindustry Technology, Da-Yeh University, Dacun, Changhua, Taiwan b Department of Health Food, Chung Chou University of Science and Technology, Yuanlin, Changhua, Taiwan c Department of Biomedical Sciences, Chung Shan Medical University, Taichung City, Taiwan article info abstract Article history: In this study, leaves and stems of Talinum triangulare were sequentially extracted with Received 19 April 2014 phosphate buffer solution to obtain PTL and PTS (phosphate buffered extracts of T. trian- Received in revised form gulare leaves and stems), with 75% ethanol to obtained ETL and ETS (ethanol extracts of T. 14 July 2014 triangulare leaves and stems), or with 90C boiling water to obtain WTL and WTS (water Accepted 17 July 2014 extracts of T. triangulare leaves and stems). We investigated the antioxidant activities of Available online 5 January 2015 various T. triangulare extracts, analyzed the extracts' stimulations on human mononuclear cell (MNC) growth and secretion of cytokines (interleukin-1 beta, interferon-g, and tumor Keywords: necrosis factor-a) and nitric oxide, and then assayed their subsequent inhibitions on human antioxidant activities leukemic U937 cell growth. Results indicated that extracts of T. triangulare showed significant human leukemic U937 cells antioxidant activities. Among these extracts, WTS showed the highest stimulatory effect on human mononuclear cells human MNC growth. The secretion levels of interleukin-1 beta, interferon-g, and tumor immunomodulatory activity necrosis factor-a in the conditioned medium, wherein human MNC was treated with 500 mg/ Talinum triangulare extracts mL WTS for 72 hours, were 1275, 859, and 2222 pg/mL, respectively. All conditioned media obtained from human MNCs cultured with various T. triangulare extracts showed significant inhibition against U937 cell growth of over 40%. These results suggest that T. triangulare extracts may be used in health foods for their immunomodulatory potential. Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. Open access under CC BY-NC-ND license. America, and much of South America. Its common names 1. Introduction include Waterleaf, Cariru, Surinam purslane, Philippine spinach, Ceylon spinach, Florida spinach, Potherb fame Talinum triangulare (Jacq.) is also called Talinum fruticosum (L.) flower, Lagos bologi, and Sweetheart [1e3]. T. triangulare is Juss. It is native plant in Mexico, the Caribbean, the Central widely grown in tropical regions as a leaf vegetable. * Corresponding author. Department of Bioindustry Technology, Da-Yeh University, Number 168, University Road, Dacun, Changhua 51591, Taiwan. E-mail address: [email protected] (M.S. Tsai). http://dx.doi.org/10.1016/j.jfda.2014.07.010 1021-9498/Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. Open access under CC BY-NC-ND license. journal of food and drug analysis 23 (2015) 294e302 295 Originating from the tropical Africa as a terrestrial herbaceous (water extracts of T. triangulare leaves and stems, respec- plant, it is now widely cultivated as a medicinal and food crop tively). All samples were stored at À80C until use. in India, South America, other parts of Asia, and Nigeria [4e6]. Several articles have shown that T. triangulare can increase 2.1.2. Reagents stamina and function as an immunostimulant [7,8].In Roswell Park Memorial Institute (RPMI) 1640 and fetal bovine Taiwan, T. triangulare has been used in the treatment and serum (FBS) were purchased from Hyclone (Logan, UT, USA). prevention of hepatic ailments and cancer in folk medicine. Sodium bicarbonate, HEPES, L-glutamine, and sodium pyruvate Some vegetable extracts have been found to exhibit func- were purchased from Sigma Chemical Co. (St. Louis, MO, USA). tional uses; in certain cases, they function as antioxidants Trypan blue was obtained from e-Bioscience (San Diego, CA, e [5,9 12], angiotensin I-converting enzyme inhibitors [13,14], USA). Potassium ferricyanide (K3Fe(CN)6), Trolox, a,a-diphenyl- antibiotics [15], and cancer cells' inhibitors [16e18]. Many b-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzthiazoline-6- human diseases are known to be related to free radicals. The sulfonate) (ABTS), EDTA, and a-tocopherol were also purchased free radical scavenging medicines are antioxidants in nature. from Sigma Chemical Co. Hence, a relationship exists between human health and minor nutrients exhibiting antioxidant activities, such as vitamin C, 2.1.3. Flavonoids and phenolic acid contents in different T. vitamin E, b-carotene, flavonoids, and other antioxidants [19]. triangulare extracts Furthermore, numerous studies (epidemiological, case- T. triangulare extracts were analyzed for flavonoids and control, or prospective and retrospective cohort) related to phenolic acid compositions via high-performance liquid dietary antioxidant intake have been linked to reducing can- chromatography (L-2130; Hitachi Co., katsuda, Japan) using an cer risks [17,20]. RP-18GP250 column (L, 250 mm; ID, 4.6 mm) and UV/Vis de- In investigating antioxidant activities, studies on vegeta- tectors. Flavonoids and phenolic acid were detected at 280 nm bles' effects in immunomodulation and cancer-cell-growth and separated using a linear gradient elution program with a inhibition may also yield a deeper insight into their func- mobile phase containing solvent A (10% methanol with 0.05% tionality. Vegetable extracts with high antioxidant activities formic acid) and solvent B (70% methanol with 0.05% formic can also be used for food preservation. Therefore, in this acid). The gradient program is shown in Table S1. The flow study, we investigated the extracts of T. triangulare for their rate used was set at 1.0 mL/min throughout the gradient. antioxidant activities as well as their inhibitory effects on the Identification and quantification were accomplished by growth of human leukemic U937 cells, by using an indirect comparing the retention time of peaks in the methanol- model and studying their immunomodulatory activities. containing solutions to that of the standard compounds. 2.2. Antioxidant activities 2. Materials and methods 2.2.1. Reducing power 2.1. Materials A method developed by Chiang and Chang [19] for a reducing power test was used. In brief, sample solutions, a-tocopherol, 2.1.1. T. triangulare samples and butylated hydroxyanisole methanolic solutions (positive Fresh T. triangulare was obtained from Chungliao (Nantou, control group) were spiked with 2.5 mL of phosphate buffer Taiwan). T. triangulare samples were separated into leaf and and 2.5 mL of 1% potassium ferricyanide. Mixtures were kept stem, cleaned and immediately stored at À20C overnight, in a 50C water bath for 20 minutes, cooled by placing it in 20C then lyophilized for 48 hours. Following lyophilization, the water bath for 5 minutes, spiked with 2.5 mL of 10% tri- dried samples were crushed to 30 mesh powders. The dried chloroacetic acid, and then centrifuged at 800g for 10 minutes. powders were stored at À20C in darkness. The supernatant (5 mL) was mixed with 5 mL of distilled water The extracts were prepared using the methods described and 1 mL of 0.1% ferric chloride. The absorbance at 700 nm by Nagai and Inoue [21] and Guo et al [22] with modifications. was then detected with a spectrophotometer after reaction for Three grams of leaf and stem powders was suspended and 10 minutes; higher absorbance (A700) represents stronger extracted with 30 volumes of 10 mM of sodium phosphate reducing power. buffer (pH 7.0) then shaken at 4C for 24 hours. Extracts were centrifuged at 4000g for 30 minutes, and the supernatants 2.2.2. DPPH radical scavenging activity were pooled, lyophilized, and denoted as PTL (phosphate The scavenging effect on DPPH free radical was measured buffered extract of T. triangulare leaf) or PTS (phosphate buff- following the method described by Shimada et al [23] with ered extract of T. triangulare stem). The residues were extrac- some modifications. Sample solutions (5 mL) and a-tocopherol ted with 30 mL 75% ethanol at 4C for 24 hours. Extracts were methanolic solutions (positive control group) were added to centrifuged at 4000g for 30 minutes, and the supernatants 1 mL of 1 mM DPPH in methanolic solution. The mixture was were pooled, lyophilized, and denoted as ETL (ethanol extract shaken and left to stand for 30 minutes at room temperature. of T. triangulare leaves) and ETS (ethanol extract of T. triangu- The absorbance rates of the resulting solution and the positive lare stems). Three grams of leaf and stem powders was control group was measured at 517 nm. The lower absorbance extracted with distilled water at 90 C for 2 hours. The extracts (A517) represents a higher DPPH scavenging activity, which is were centrifuged at 4000g for 30 minutes, and the superna- expressed as [1 e (test sample absorbance/blank sample tants were pooled, lyophilized, and denoted as WTL and WTS absorbance)] Â 100%. 296 journal of food and drug analysis 23 (2015) 294e302 2.2.3. Ferrous ion chelating ability filtered to obtain condition media (CMs). The concentrations of The method described by Decker and Welch [24] was adopted. cytokines including interleukin-1 beta (IL-1b), interferon- Five milliliters of the test solutions, including sample and gamma (IFN-g), and tumor necrosis factor-alpha (TNF-a)were EDTA solutions, was spiked with 0.1 mL of 2mM FeCl2 and determined using commercial enzyme-linked immunoassay 0.2 mL of 5mM ferrozine solutions. After reaction for 10 mi- kits (Bender MedSystem, Inc., Burlingame, CA, USA).